DETERMINATION OF AFLATOXIN M1 IN RAW COW'S MILK BY USING HPLC- FLD, IN INJIBARA TOWN, AWI ZONE, AMHARA, ETHIOPIA

The aim of this study, therefore, provides information about Aflatoxin levels in raw cow’s milk in Injibara Town of Awi Administrative zone by using HPLC-FLD. A good linearity of standard calibration was found for AFM1 at a range of 0.5–7 µg/L. Regression coefficient (R2) values were 0.9999, whereas slope and intercept were 2.5278 and 0.1012, respectively. The average recoveries for the spiked samples were range from76.62– 90.98 % and the RSD values ranged between 2.55–7.36 %. The results showed that 15 % of samples (3 out of 20) were contaminated with AFM1 in the range of 0.046–0.22 µg/L. The average contamination level was 0.121 µg/L. The determined mean values of AFM1 in the collected milk samples were above the standard limit of the European Commission and lower than the level established by United States regulations. Further monitoring of Aflatoxin in milk samples from different regions of the country is justified to conclusively determine the actual safe/risks and possibly low Aflatoxins-risk milk production areas.


Introduction
Generally, nutrition that found highly in the human body is milk and its products. In developed countries like Europe, the amount of milk and its products consumed per day is approximately 340 g/day per person [1]. Milk can be used in all stages of human beings, especially for mothers, children, old people, and infants and it is the basic dietary for human beings [2,3]. From the different types of mycotoxins, Aflatoxin is the main one which mainly found in milk that generated from the animal feed by the toxigenic materials mainly by fungi such as Aspergillus flavus, Aspergillus parasiticus and the rare Aspergillus nomius [4].
Generally, the word of "Aflatoxin" is derived from the three words; one is "a" from the Aspergillus genus and the other is "fla" from the species flavus and the other third is from toxin which means poison. Different scholars stated that ingestion is the main root of Aflatoxin species for living things, especially for humans and animals through the feeding channel [5]. From the different types of Aflatoxins, such as AFB1, AFB2, AFG1, AFG2, AFB1 and AFM2, Aflatoxin M1 (AFM1) is the most common [6].
The bloodstream contains the byproduct of the metabolisms and in the bile or urine which is excreted, as well as mainly, Aflatoxin M1released in milk and eggs [7,8]. Some of the Aflatoxin is metabolized to the Aflatoxin M1 in the cattle when they ingested the Aflatoxin B1 and which is found and excreted in the milk. Aflatoxin (M1) can be found in the milk product such as cheese and yogurt because of this Aflatoxin is thermally stable in the process of pasteurization/thermal treatments of milk [7,9]. In the case of parts of milk, Aflatoxin M1 is strongly attached due to the production of this Aflatoxin M1 during the process of the technological generation of cheese. Several parameters that determine the contents of Aflatoxin M1 in the milk and from these factors, some of them are the breeding system of animals, period of location, infections of mammary, etc. After a cattle is ingested the AFB1, AFM1 is within one (12-24 hrs) detected and within a short period, it can reach a high level. The aim of this study, therefore, will provide information about Aflatoxin levels in raw cow's milk in Injibara Town of Awi Administrative zone by using HPLC-FLD method detection. 4

Description of the study site
Amhara is one of the regional states of Ethiopia in which located between 8°45՛N and 13°45՛N latitude and 35°46՛E and 40°25՛E longitude in North West Ethiopia. The climate of the Amhara Region is affected significantly by variation in altitude, latitudinal position, prevailing winds, air pressure and circulation and its proximity to the sea [10]. The study was conducted in Injibara Town of Awi zone; one zone of Amhara regional state. The study was conducted from February to August, 2018/2019. Awi is the one among the 11 zone of the Amhara regional state. The highest and lowest altitude of Injibara is recorded to be 2540 m.a.s.l and 3000 m.a.s.l respectively. According to the town's Administration, the total area of the town is estimated to be 28.3 km. It was divided into 5 urban Kebeles under the town administration. According to the development plan of Injibara, social and personal services are the dominant sector in the town's economy followed by trade (especially charcoal trade), tourism and Manufacturing dominating sector providing employment.

Sampling method and sample collection
A total of 20 raw cow's milk (500 mL each) samples were collected from individual farmers at Injibara Town of Awi Zone. Each sample was collected at night and morning during the day, through a systematic random sampling method and the collected raw cow's milk samples were 5 placed in a clear polyethylene bag. The raw caw's milk was kept on the ice during the transportation and the collected sample was stored at -18 °C in the refrigerator at Laboratory until the analysis.

Apparatus and Instruments
The following equipment was used in this study: electronic analytical balance was used for weighing standard, fast flow filter paper and funnel was used to filter the milk samples, micropipettes (100, 250, 500, 1000 mL) were used for measuring volume of standard solutions, volumetric flasks (50, 100, 250, 500, 1000 mL) were also used for preparing working standard solutions and stock solutions, 2 mL autosampler vial with cap was used for stored the sample until analysis in HPLC-FLD, Nitrogen gas (purity > 99.8 %) was used for evaporated the filtrate, Syringe filters 0.45 µm used for sample filtration after filter paper, Immune-affinity column used to separate Aflatoxins M1 from the mixture of solution, chromatographic Eclipsed plus column C18 length of 100 mm internal diameter 4.6 mm, particle diameter of packing material 5 µm and HPLC equipped with FLD capable of providing about 365 nm excitation and 435 nm emission λ were used to analysis Aflatoxins M1 in milk samples.

Experimental Procedures
The procedure used for AFM1 extraction and clean-up was based on the instructions described by Ethiopian confirmative assessment enterprise agency (ECAE) for AFM1 determination in

Preparation of milk sample and Extraction
The freeze samples were warmed in a water bath with 37 °C for 30 min and 25 mL of prepared sample was added into a 50 ml falcon tube and it was centrifuge with 2000 g for 5 min. Then the solution was filtered by fast flow (541) filter paper that attached with the immune-affinity column and the filtrate was passed in the immune-affinity column with 1drop/sec. Then the immune-affinity column was washed with 5 ml of 10 mM PBS three times and dries by passing air through it. Aflatoxin M1 was eluted with 1 mL of methanol three times and filter through 0.45 µm syringe filter into amber auto-sampler and transfer to 2 mL vial for HPLC analysis.

Method Validation
The proposed method was validated by evaluating different parameters as a limit of detection (LOD), limit of quantitation (LOQ), accuracy (in terms of recovery) and precision (in terms of repeatability) [11].

Limit of Detection
Limit of detection (LOD) is the minimum concentration of analyte that can be detected but not necessarily quantified with acceptable uncertainty. Limit of detection for each Aflatoxin M1 was determined from an analysis of three replicates of method blanks [12]. LOD was calculated as: Where, Sbl is the standard deviation of the method blank.

Limit of Quantification
Limit of quantification (LOQ) is the lowest concentration of analyte that can be determined with an acceptable level of uncertainty. Limit of quantification was obtained from an analysis of three replicate of method blanks. The limit of quantification was calculated as ten times the standard deviation of the blank. Where, Sbl is the standard deviation of the method blank [12].

Precision and Accuracy
Precision is the extent of the consistency of results as they are obtained during repeated applications a specified determination method. It was evaluated regarding repeatability by estimating the relative standard deviation (RSD) of the recovery percentage for each spiked The percentage recoveries of the analyte were calculated to evaluate the accuracy of the analytical procedure. Recovery was then calculated as: -. and 435 nm, respectively.

Statistical analysis
All statistical analyses were done by the Microsoft Office Excel-2010 program. Data expressed as mean ± standard deviation (SD) of three replicate experiments for recovery. calibration curve was plotted with six points for each standard using peak area against concentrations and the calibration curve is given in Figure 2. Linearity of HPLC-FLD system was done by injecting different concentrations of standard and the system was calibrated by using the working solutions of Aflatoxin M1 in the range of 0.5-7 µg/L and the response (peak area) is given in Table 1.    Table 2, the limit of detection (LOD) and limit of quantification (LOQ) values for AFM1 analyzed in the milk samples were 0.045 and1.35 ug/L, respectively.

Accuracy and Precision
The results of accuracy and precision were evaluated through recovery tests. Accuracy of the method was determined by matrix spike recovery studies and precision was expressed as relative standard deviation (RSD) of triplicate results. In this study, the recovery test was done by spiking a suitable known quantity of Aflatoxin M1 standard solution into a test portion of the sample.
The percentage recoveries of raw cow's milk samples were given in Table 3. As can be seen from    In Table 4 summarizes, the number of samples analyzed and the number of samples found to contain detectable levels of AFM1 contamination in raw cow's milk. In this study that 2(10 %) of the examined raw milk samples from 20 samples were contaminated with Aflatoxin M1 and exceeded the European Union limit (0.05 μg/L). The variations in AFM1 levels among studies/sites could be associated with different reasons such as season, feeding systems, farm management practices, and analytical methods. It could also be linked to the carryover rate of AFB1 as AFM1 in milk which varies widely among animals, days and from one milking session to the next and it is greatly influenced by physiological factors such as diet and health status of animals [14]. Other studies from urban centers in Kenya have been reported AFM1 levels up to 0.68 µg/L. In contrast to this result, the level of AFM1 contamination found in raw milk collected from Khartoum state in Sudan, with an average concentration of 2.07µg/L and a maximum of 6.9 µg/L was higher than what was found in this study. Egypt 0.050 -Low [18] Low = within the EU standard (0.05 μg/L); Medium = Within the USA standard (0.5 μg/L) The quality of milk in terms of Aflatoxin M1 contamination in this study was compared with other countries ( Table 5) and the AFM1 contamination level was slightly higher than the AFM1 of milk studied in Egypt (0.05 μg/L) [18]. However, the quality of milk in terms of the level of AFM1 in milk was lower than the milk samples reported from India [15][16][17]. 12 The difference of AFM1 in milk in Ethiopia, Egypt and Iran could be due to the difference in feed type used in Ethiopia which is mainly by-products wheat as compared to Ethiopia, Egypt and Iran mainly used by-products of maize and groundnut feeds that are the most susceptible commodities to Aflatoxin contamination as compared wheat [19,20]. It is interesting to note that the Aflatoxin level of milk in this study was in the " Medium" category (within the Stringent USA standard) similar to most reports from different countries, especially from India [21,15,16] ( Table 5).
The Chromatogram of tested samples is given below in Figure 3: Retention time (minutes) and peak area in X and Y-axis values.

Conclusions and recommendations 4.1 Conclusion
 Milk is an essential source of nutrition and thus its consumption is increasing parallel to the continuous increase of the human population. Therefore, raw caw's milk has to be controlled continuously for the presence of AFM1 contamination. It is extremely important to maintain low levels of AFM1. Efficient control of AFM1 in milk requires efficient and easily performed analytical methods. According to results obtained in this study, seventeen(17) of the milk samples were no detected for Aflatoxin M1 contamination; whereas one sample below the detection limit and the remaining three from 20 samples were within the stringent USA level of 0.5 μg/L. The AFM1 level of milk in this study was in the "Medium category" which was comparable to those AFM1 levels obtained elsewhere. It is important to prevent toxin production in feed, as well as create effective detoxification processes thus further studies are required to measure the level of AFM1 in the rations of supplementary feeds given to lactating cows concerning the level of AFM1 in milk.
 Implementing a food supervision control system in the dairy products industries and Funding: This study was supported by fundamental research funds for the Injibara University, Ethiopia.